This invention relates generally to the fields of molecular genetics and plant breeding. More particularly, it relates to a means of moving novel, stably inherited, variant forms of DNA into maize (Zea mays L.), also referred to as corn in the United States. These novel DNA sequences, derived from intergeneric hybridization between Eastern gamagrass (Tripsacum dactyloides L.) and perennial teosinte (Zea diploperennis Iltis, Doebley and Guzmxc3xa1n), provide unique markers for assisting selection of desirable traits in plant breeding programs, for detection of target DNA sequences in genetic analyses, and for the identification of new genes for corn improvement that may enhance resistance to insect pests and diseases, drought stress tolerance, cold tolerance, perennialism, grain yield, totipotency, apomixis, improved root systems, tolerance of water-logged soils, tolerance of high-aluminum and acidic soils, improved grain quality, enhanced forage quality, and adaptability to a CO2 enriched atmosphere.
Molecular Genetics. Genetics is the study of genes and heritable traits in biological organisms. In plant breeding, the goal of molecular genetics is to identify genes that confer desired traits to crop plants, and to use molecular markers (DNA signposts that are closely associated with specific genes) to identify individuals that carry the gene or genes of interest in plants (Morris 1998), to determine the DNA sequences and characterize gene expression and function. A genetic marker is a variant allele that is used to label a biological structure or process throughout the course of an experiment. Variants in DNA and proteins are used as markers in molecular genetics. Genetic analysis of molecular variants can identify a particular gene that is important for a biological process. Mutation is the process whereby nucleotide sequences and genes change from the reference form generally designated wild type to a different form, and mutants are the source of variant genotypes in genetic analysis that allow selection of new phenotypes (Griffiths et al. 1993). Mutations occur at the level of a specified nucleotide sequence, the gene (i.e. DNA sequence), or the chromosome (i.e. the hereditary package in which units of DNA containing specific nucleotide sequences and genes are supercoiled with proteins). In a genetic mutation, the nucleotides that comprise the wild type allele of a gene (i.e. reference form that exists at a particular locus) is altered. In chromosome mutations, segments of chromosomes, whole chromosomes, or entire sets of chromosomes change via inversion, translocation, fusions and deletions.
In general, mutations are very rare, and most newly formed mutations are deleterious. Data on mutation frequencies for seven genes in maize provides a baseline indicating the rarity of mutations in maize (Stadler 1951). Mutation frequency ranged from 0.000492% (i.e. 492 mutants out of a million gametes) in the red color(R) gene; 0.000106% (i.e. 106 out of a million) for the inhibitor of R (I) gene; 0.000011% (i.e. 11 out of a million) for the purple aleurone (Pr) gene; 0.0000024% (i.e. 2.4 out of a million) for the starchy (Su) gene; 0.0000022% (i.e. 2.2 out of a million) for the yellow color (Y) gene; 0.0000012% (i.e. 1.2 out of a million) for the normal kernel (Sh) gene, and 0% (i.e. 0 out of a million for the waxy gene (Wx).
Because spontaneous mutations are rare, geneticists and plant breeders typically use mutagens (i.e. agents such as chemicals and radiation to increase the frequency of mutation rates) to obtain variant forms that can be used in genetic analysis and selection of new varieties. Another method of inducing mutagenesis in maize is transposon tagging whereby a maize line is crossed with a line containing one of the three systems of transposable elements found in maize. When a transposable element inserts into a gene, it causes a mutation. The reported mutation frequencies for transposable element mutator lines varies from 1 in a thousand to 1 in a million (Chomet 1994). To find a mutation using one of these mutagenic lines, a breeder must screen a minimum of 100,000 progeny.
Plant Breeding. Conventional plant breeding is the science that utilizes crosses between individuals with different genetic constitutions. The resulting recombination of genes between different lines, families, species, or genera produces new hybrids from which desirable traits are selected. Plant breeding is achieved by controlling reproduction. Since maize is a sexually reproducing plant, techniques for controlled pollination are frequently employed to obtain new hybrids. Controlling reproduction in maize involves continually repeating two basic procedures: (1) evaluating a series of genotypes, and (2) self-pollinating or crossing among the most superior plants to obtain the next generation of genotypes or progeny. Controlled pollinations in maize utilize two procedures: (1) detasseling, and (2) hand pollination.
Maize is a monoecious grass that has separate male and female flowers on the same plant. The male or staminate flowers produce pollen in the tassel at the apex of the maize stalk, and the female or pistillate flowers that produce the grain when pollinated are borne laterally in leaf axils tangential to the stalk. Pollination is accomplished by transfer of pollen from the tassel to silks which emerge from the axillary pistillate ears. Since maize is wind-pollinated, controlled pollination in which pollen collected from the tassel of one plant and transferred by hand to the silks of the same or another plant, is a technique used in maize breeding. The steps involved in making controlled crosses and self-pollinations in maize are standard practice (Neuffer 1982) and are as follows: (1) the ear emerging from the leaf shoot is covered with an ear shoot bag one or two days before the silks emerge to prevent contamination by stray pollen; (2) prior to making a pollination, the ear shoot bag is quickly removed and the silks cut with a knife to form a short brush, then the bag is immediately placed back over the ear; (3) also prior to making a pollination, the tassel is covered with a tassel bag to collect pollen; (3) on the day crosses are made, the tassel bag with the desired pollen is carried to the plant for crossing, the ear shoot bag is removed and the pollen dusted on the silk brush, the tassel bag is then fastened in place over the pollinated shoot to protect the developing ear.
Zea diploperennis (hereafter referred to as diploperennis), is a diploid perennial teosinte and a wild relative of maize endemic to the mountains of Jalisco, Mexico. Diploperennis is in the same genus as maize, has the same chromosome number (2n=20), and can hybridize naturally with it.
Tripsacum is a polyploid, rhizomatous perennial grass that is a more distant wild relative of maize and has a different chromosome number (x=18, 2n=36 or 2n=72). Tripsacum is not know to naturally form fertile hybrids with maize or the wild Zeas. The progeny of (maize X Tripsacum) obtained by artificial methods have ten maize chromosomes and either 18 or 36 Tripsacum chromosomes and are male sterile. Female fertility can be partially restored using special techniques that eliminate most of the Tripsacum chromosomes (Mangelsdorf 1974). Plants obtained by crossing Tripsacum and maize (Zea mays L.) employing Tripsacum as the pollen donor have unreduced gametes with a complete set of Zea chromosomes and a complete set of Tripsacum chromosomes. There is one report of a successful reciprocal cross in which Tripsacum was pollinated by maize that required embryo culture techniques to bring the embryo to maturity, and the plants were sterile (Farquharson 1957). Maize-Tripsacum hybrids have been crossed with teosinte to created a trigenomic hybrid that has a total of 38 chromosomes; 10 from maize, 18 from Tripsacum and 10 from teosinte. The resulting trigenomic plants were all male sterile and had a high degree of female infertility (Mangelsdorf 1974; Galinat 1986).
Based on known crossability relationships between Zea and Tripsacum and the results of prior crosses between them, the success of the crosses between Zea diploperennis and Tripsacum resulting in viable, fully fertile plants with chromosome numbers of 2n=20 (Eubanks 1995, 1997) could not have been predicted. Reduction in chromosome number in the interspecific crosses was unexpected based on prior art. The fertility of plants resulting from the cross made both ways with Tripsacum as pollen donor and pollen recipient was also unexpected based on prior art.
Although the base chromosome numbers of Tripsacum and Zea diploperennis are different, x=10 in Zea and x=18 in Tripsacum, their respective total chromosome lengths are almost equal. The total length of the 18 Tripsacum dactyloides chromosomes is 492.5xcexc (Chandravadana et al. 1971), and the total length of the 10 Zea diploperennis chromosomes is 501.64xcexc (Pasupuleti and Galinat 1982). It is not easy to obtain a hybrid plant when crossing Tripsacum and diploperennis. Hundreds of pollinations are required to obtain a viable seed, and approximately half of seedlings that germinate die soon after germination. However, as evidenced by cross fertility and chromosome number, when precise alignments occur between homologous regions of the chromosomes of Tripsacum and diploperennis there is a sufficient degree of pairing to occasionally enable the rare and unexpected success of this cross.
The unexpected fertility of Tripsacum-perennial teosinte hybrids, and their cross-fertility with maize, are of great value because they provide opportunity for directly crossing the recombined intergeneric germplasm with maize. In addition to providing a genetic bridge for importing Tripsacum genes into maize, Tripsacum-diploperennis hybrids provide a mechanism for importing any new Tripsacum genes not found in maize or the wild Zeas, and any de novo genetic material that arises from these wide species crosses into maize using traditional plant breeding techniques.
DNA fingerprinting has revealed that new Tripsacum alleles not found in maize or the wild Zeas and de novo sequences newly created via the wide cross are stably inherited in the progeny of succeeding generations and can be conferred to maize by crossing maize with Tripsacum-diploperennis containing de novo nucleotide sequences and alleles unique to Tripsacum. For purposes herein, de novo genetic material refers to regions where new allelic forms of DNA sequences are repeatedly and reliably created whenever crosses between Tripsacum and Zea diploperennis produce viable, fertile plants.
Feasibility has been demonstrated in plants derived from crossing Tripsacum-diploperennis with maize that exhibit resistance to western corn rootworm (Diabrotica virgifera Le Conte) and corn borer, tolerance to drought, and have properties of perennialism. Investigation and characterization of association with other traits such as response to high levels of atmospheric CO2 are in process.
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In one embodiment of the invention, there is provided a method for conferring novel genetic materials into maize. In the first step of the method, a Tripsacum plant is pollinated by pollen from a perennial teosinte plant by controlled pollination technique, or vice versa, a perennial teosinte plant is pollinated by pollen from a Tripsacum dactyloides plant. The resulting intergeneric hybrids derived in step 1 are fully fertile and cross-fertile with maize. The hybrid plants are characterized by their utility as a genetic bridge to transfer novel genetic materials into maize and their unexpected chromosome number of 2n=20 instead of the expected 2n=28 or 2n=46 if a full, unmodified complement of perennial teosinte haploid chromosomes (n=10) and diploid Tripsacum (n=18) or tetraploid Tripsacum (n=36) chromosomes were transmitted to the resulting hybrid progeny.
In another embodiment of the invention, in step 2 of the method, the intergeneric hybrid plant (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum) is crossed with maize by controlled pollination. In the cross, the pollen of (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum) is transferred to maize silks, or maize pollen is transferred to the silks of (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum). This invention relates to hybrid seed, hybrid plants produced by the seed and/or tissue culture, variants, mutants, modifications, and cellular and molecular components of the hybrid plants that contain novel genetic materials derived from (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum).
In another embodiment of the invention, in step 3 of the method, the trigeneric hybrid plant obtained from crossing (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum) with maize by controlled pollination as described in step 2, is backcrossed to maize or (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum). In the backcross, the pollen of the trigeneric hybrid plant is transferred to the silks of one of the original parents (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum) or maize. This invention relates to hybrid seed, hybrid plants produced by the seed and/or tissue culture, variants, mutants, modifications, and cellular and molecular components of the backcrossed hybrid plants that contain novel genetic materials derived from (Tripsacum X perennial teosinte) or (perennial teosinte X Tripsacum).
In another embodiment of the invention, there is provided plants and plant tissues produced by the method of crossing maize with a Tripsacum-diploperennis hybrid that contain novel genetic materials and exhibit beneficial agronomic traits. For example, these plants may contain novel genes for such traits as pest and pathogen resistance, drought tolerance, cold tolerance, water-logging tolerance, improved grain quality, improved forage quality, totipotency, perennialism, tolerance to acidic soils, tolerance to high-aluminum soils, enhanced adaptability in a carbon dioxide enriched environment and can be employed in recurrent selection breeding programs to select for hybrid maize that exhibit such traits.
For the purposes of this application, the following terms are defined to provide a clear and consistent description of the invention.
Allele. One of the different forms of a gene that can exist at a single locus.
Autoradiography. A process in which radioactive materials are incorporated into cellular components, then placed next to a film or photographic emulsion to produce patterns on the film that correspond to the location of the radioactive compounds within the cell.
Electrophoresis. A technique for separating the components of a mixture of molecules (proteins, DNAs, or RNAs) in an electric field within a gel matrix.
Genetic markers. Alleles used as experimental probes to keep track of an individual, a tissue, a cell, a nucleus, a chromosome, or a gene.
Gene. The fundamental physical and functional unit of heredity that carries information from one generation to the next. The plant gene is xe2x80x9ca DNA sequence of which a segment is regularly or conditionally transcribed at some time in either or both generations of the plant. The DNA is understood to include not only the exons and introns of the structural gene but the cis 5xe2x80x2 and 3xe2x80x2 regions in which a sequence change can affect gene expressionxe2x80x9d (Neuffer, Coe and Wessler 1997).
Genotype. The allelic composition of a cellxe2x80x94either of the entire cell or, more commonly, for a certain gene or a set of genes of an individual.
Hybrid plant. An individual plant produced by crossing two parents of different genotypes or germplasm backgrounds.
Locus. The place on a chromosome where a gene is located.
Molecular genetics. The study of the molecular processes underlying gene structure and function.
Mutagen. An agent that is capable of increasing the mutation rate.
Mutation. (1) The process that produces nucleotide sequences, genes, genetic elements, or chromosomes differing from the wild-type. (2) The nucleotide sequences, genes, genetic elements, or chromosomes that result from such a process.
Plant breeding. The application of genetic analysis to development of plant lines better suited for human purposes.
Probe. Defined nucleic acid segment that can be used to identify specific molecules bearing the complementary DNA or RNA sequence, usually through autoradiography.
Restriction enzyme. An endonuclease that will recognize specific target nucleotide sequences in DNA and cut the DNA at these points; a variety of these enzymes are known and they are extensively used in genetic engineering.
RFLP. Refers to restriction fragment length polymorphism that is a specific DNA sequence revealed as a band of particular molecular weight size on a Southern blot probed with a radiolabelled RFLP probe and is considered to be an allele of a gene.
Southern blot. Transfer of electrophoretically separated fragments of DNA from the gel to an absorbent surface such as paper or a membrane which is then immersed in a solution containing a labeled probe that will bind to homologous DNA sequences.
Totipotency. The ability of a cell to proceed through all the stages of development and thus produce a normal adult.
Wild typexe2x80x94refers to a reference and it can mean an organism, set of genes, gene or nucleotide sequence. For purposes herein the wild type refers to the parents of hybrid progeny.